6 alpha, 21-difluoro-16 alpha-hydroxy-steroids



United States Patent D 2,838,547 6oc,2l-DIFLUORO-16ot-HYDROXY-STEROIDSBarney J. Magerlein, William P. Schneider, Oldrich K.

Sebek, and George B. Spero, Kalamazoo, Mich., assignors to The UpjohnCompany, Kalamazoo, Micln, a corporation of Michigan No Drawing.Application February 19, 1958 Serial No. 716,015;

19 Claims. cum-397.45

The present invention relates to steroid compounds and is moreparticularly concerned with 6a,2l-difluoro- 11,8,16u,l7a trihydroxy 4pregnene 3,20 dione, Y

6a.,9a,21 trifiuoro 11;8,l6a,17a-trihydroxy 4 preg-1,4-pregnadiene-3,ZO-dione, 6a,9u,21-trifluorol13,161,17a-trihydroxy-l,4-pregnadiene-3,ZO-dione, the 16-esters thereof, thell-keto analogues and the 16 esters thereof, and a method for theproduction thereof.

The novel compounds of this invention are illustratively represented bythe following formula:

and R is selected from the group consisting of hydrogen and the acylradical of an organic carboxylic acid, preferably a hydrocarboncarboxylic acid containing from one to twelve carbon atoms, inclusive.

The new compounds,6a,21-difluoro-11p,16a,17a-trihydroXy-4-pregnene-3,20adione,6u,9a,21-trifluoro-1lfl, 16a,l7u-trihydroxy-4-pregnene-3,20-dione,6u,21-difluoro- 1 118,1604, l7a-trihydroxy-1,4-pregnadiene-3,ZO-dione,6oz, 9:1,2 l-trifluoro-l 1 8, 16a, l7ot-trihydro'xy- 1,4-pregnadiene-3,20-dione, their 16 esters and the ll-keto analogues and the estersthereof, are highly active adrenocortical hormones having greaterglucocorticoid and anti-inflammatory activity than hydrocortisone orcortisone. In addition these compounds have diuretic activity and havesaltlosing properties which make them especialy well suited in themanagement of chronic congestive heart failure and in the treatment ofcirrhosis of the liver, the nephrotic and adrenogenital syndromes andthe treatment of eclampsia and preeclampsia.

droxy4-pregnene-3,20-dione,6a,21-difluoro-11B,16a,17atrihydroxy-l,4-pregnadiene-30,20-dione,6a,9ot,21-t1ifll.l0- ro-l18,16a,17a-trihydroxy-1,4-pregnadiene-3,ZO-dione, the

l6 esters thereof, the ll-keto analogues and the 16-esters thereof canbe given in oral, parenteral 0r topical compositions. The compounds canbe administered to the animal organism in conventional dosage forms suchas pills, tablets and capsules for oral use or in conventional liquidforms as are used with natural and synthetic cortical steroid hormonesfor injection use. For topical use they can be administered in the formof ointments, creams, lotions and the like with or without co-actingantibiotics, germicides and the like. i i

The process of the present invention comprises microbiologicalhydroxylation of 6a,2l-diflu0ro-11;3,17a-dihy-. droxy-4 pregnene,6a,9x-,21-triflu0rO-115,17o1-dlhYd1'OXY 4-pregnene-3,20-dione and the n-analogues thereof to produce the corresponding l6-hydroxy compounds.Esterification of the thus produced 16-hydroxylated compounds isproductive of the 16-esters. -The corresponding ll-keto analogues ofthese 16-hydroxylated' compounds are obtained by oxidation of thell-hydroxyl group of the above 16-esterified compounds with an oxidationagent such as chromic acid. If the free alcohols of the ll-ketocompounds are desired, the additional step of hydrolyzing the 16-esterssuch as with an alkali metal base is necessary.

Starting materials for the present invention are 60:,21- difiuoro115,17a dihydroxy-4-pregnene-3,20-dione, 6oz, ,2lt1ifi1101'O-1113,17a-dihydroxy-4-pregnene-3,ZO-dione, 6a,2 l-difluoro-l1B,17u-dihydroxy-1,4-pregnadiene-3,20-dione, and6a,9a,21-trifluoro-11,8,l7ot-dihydroxy-L4-pregnadiene-3,20-dione,prepared as shown in the preparations.

In the bioconversion step of the present invention, the operationalconditions and reaction procedure and details may be those already knownin the art of steroid bioconversion as illustrated by the Murray et a1.U. S. Patent 2,602,769, issued July 8, 952, utilizinghowever the actionof an organism of the genus Streptomyces. Among the species which areuseful in the fermentation of steroids are Streptomyces roseochromogenus(Waksman Collection 3689), Streptomyces sp. (A. T. C. C. 11009), andStreptomyces roseochromogenus (A. T. C. C. 3347).

The selected species of actinomycete is'grown on a medium suitablycontaining assimilable non-steroid carbon, illustratively carbohydrates,such as dextrose; assimilable nitrogen, illustratively soluble orinsoluble proteins, peptones or amino acids; and mineral constituents,illustratively sodium or ammonium phosphate and magnesium sulfate.before inoculation of between about 6.5 to about 7.8 though a higher orlower pH may be used. A pH of between about 6.8 and about 7.4 ispreferred for the growth of actinomycetes and a temperature range fromabout 20 to about 35 degrees centigrade with about 20 to 32 degreescentigrade preferred.

The growth period required before the steroid to be fermented is exposedto the actinomycete does not appear to be critical, for example, thesteroid may be added either before sterilization of the medium, at thetime of inoculating the medium or at sometime later, for example, 24 or48 hours later. The addition of steroid substrate to be fermented may beaccomplished in any suitable manner, such as by dispersing the steroidsubstrate, either alone with a dispersing agent, or in solution in anorganic solvent. Either submerged or surface culture procedures may beused with facility, although submerged culture is preferred.

The temperature during the period of fermentation of the steroid may bethe same as that found suitable for the growth of the organism. It needbe maintained only within such range as supports life, active growth, orthe enzyme activity of the streptomycete.

The time required for the fermentation of the steroid varies somewhatwith the procedure. When the steroid Patented June 10, 1958' The mediummay desirably have a pH is added to the actinomycete after substantialgrowth of the organism, for example, after sixteen to 24 hours atoptimum temperature, the conversion of steroid substrate beginsimmediately and is substantially complete in from two to ten days, fivedays being generally satisfactory.

After completion of the steroid fermentation, the resuiting transformedsteroid is recovered from the fermentation reaction mixture byextracting the fermentation reaction mixture, including the fermentationliquor and mycelium with an organic solvent for steroids, for example,methyl isopropyl ketone, methylene chloride, chloroform, carbontetrachloride, ethylene chloride, trichloroethylene, ether, amylacetate, benzene, and the like. The fermentation liquor and mycelium canbe separated and then separately extracted with suitable solvents. Theextracts can be combined, either before or after washing with analkaline solution, illustratively sodium bicarbonate, suitably dried, asfor example, over anhydrous sodium sulfate, and the resulting purifiedtransformed steroid obtained by recrystallization from organic solvents,by trituration or by chromatography in order to isolate the thusobtained steroids from the other transformation products.

Bioconversion of 6a,21-difluoro-1lB,17u-dihydroxy-4- pregnene-3,20dione, 6a,9 x,21 trifiuoro-11[3,17u dihydroxy-4-pregnene-3 ,20-dione,60,2 l-difluoro-l 1B, 17e-dihydroxy-1,4-pregnadiene-3,ZO-dione, and6a,9u,2l-trifiuoro- 11B,17ct-dihydroxy-1,4-pregnadiene-3,ZO-dioneaccording to the fermentation procedure above described, is productiveof 6u,2l-difluoro-l1,6,16a,17a-trihydroxy-4-pregnene- 3,20-dione,6u,9ot,2l-trifiuoro-llfi,l6ot,l7a-trihydroxy 4- pregnene-3,20-dione,601,21 difluoro-l 1[3,16OL,170 trihydroxy-l,4-pregnadiene-3,ZO-dione,and 604,904,21-t1ifl1101'0- 11,8,16a,17a trihydroxy-1,4-pregnadiene3,20-dione, repectively.

The 16-hydroxylated compounds thus produced can be esterified to producethe corresponding l6-esters. This reaction canbe performed underesterification conditions known in'the art, e. g., by the reaction ofthe hydroxy compound with the selected acid halide, e. g., acid chlorideor acid bromide, the anhydride of a hydrocarbon carboxylic acid, or byreaction with the selected acid, in the presence of an esterificationcatalyst or with an ester under ester exchange reaction conditions.Reaction conditions which are apt to affect the labile llfl-hydroxygroup or 6-fiuoro group should be avoided. Compounds thus producedinclude the 16-acyloxy compounds represented by Formula I wherein X isfi-hydroxymethylene radical and wherein R is the acyl radical of anorganic carboxylic acid, preferably a hydrocarbon carboxylic acidcontaining from one to twelve carbon atoms, inclusive, e. g., formic,propionic, butyric, isobutyric, valeric, isovaleric, trimethylacetic,Z-methylbutyric, 3-ethylbutyric, hexanoic, diethylacetic,triethylacetic, heptanoic, octanoic, a-ethylisovaleric, succinic, acyclic acid, e. g., cyclopropylideneacetic, cyclopentylformic,cyclopentylacetic, 13- cyclohexylpropionic, cyclohexylformic,cyclohexylacetic, an aryl or alkaryl acid, e. g., benzoic, 2-, 3- or4-methylbenzoic, 2,3-, 2,4-, 2,5-, 2,6, 3,4- and 3,5-dimethylbenzoic,ethylbenzoic, 2,4,6-trimethylbenzoic, 2,4,6-triethylbenzoic,a-naphthoic, 3-methyl-a-naphthoic, an aralkyl acid, e. g., phenylacetic,phenylpropionic, diphenylacetic, triphenylacetic, an unsaturated acid,e. acrylic, maleic, vinyl acetic, propiolic, undecolic, etc.Illustrative of the esters thus produced are the l-acylates'such as60,21-difiuoro-llfl,16a,17u-trihydroxyl-pregnene-3,ZO-dione l6- acetate,6a,21-difiuoro-1lfl,16a,l7 x tri'nydroxy-1,4-pregnadiene-3,20-dioneIG-acetate, 6a,9tx,2l-trifiuoro-l15,1604,17a-trihydroxy-4-pregnene-3,20-dione 16-acetate, 611,90, ZI-trifiuoro-l1,8,16c2, 17a-trihydroxy-l,4-pregnadiene-3,20- dione 16-acetate, and thelike.

The 11 e-hydroxyl of the thus produced l6-acylate compounds can beoxidized to the corresponding 11-ketone with an oxidizing agent.Oxidizing agents such as chromic acid, potassium dichroinate,a-halo-amide, and

the like are operative. The oxidation can be carried out by a variety ofmethods, such as, for example, by oxidizing the said 11 B-hydroxysteroid in acetic acid-water solu tion with chromium trioxide, usingmolar quantities or a slight excess, such as from ten to thirty percentexcess, or by oxidizing with a haloamide or imide of an acid, such asN-bromoacetamide, N-chlorosuccinimide, or N- bromosuccinimide dissolvedin pyridine, dioxane, or other suitable solvents. At the conclusion ofthe desired oxidation reaction, the excess oxidant is generallydestroyed by addition of methyl alcohol, ethyl alcohol, and the like forthe chromic acid oxidant or a bisulfite for N-bromoacetamide,N-bromosuccinirnide and other N-haloacylamides and imides. Thereafter,the resulting ll-keto product is recovered by conventional means, suchas by dilution with water and extraction with a water-immisciblesolvent, e. g., methylene chloride, ether, benzene, toluene, ethylacetate, or the like. Illustrative of the 11- keto 1'6-acy1ates thusproduced are, for example,6c4,2ldifiuoro-16a,17a-dihydroxy-4-pregnene-3,l l,20-trione 16- acetate,60,90t,21-TifillOIO-160L,170t-dlhYdl'OXY-4-pfegn1l- 3,11,20-trione16-acetate,60,21-dlflUOIO-16Ct,17Dt-dlhydroxy-1,4-pregnadiene-3,11,20-trionel6-acetate, 6a,9a,2ltritiuoro l6oc,l7oc dihydroxy 1,4-pregnadiene3,11,20- trione 16-acetate, and the like.

The 11-keto l6-acylates thus produced may, if desired, be converted tothe free alcohols, i. e.,6a,2l-difiuoro-16a,l7a-dihydroxy-4-pregnen-3,11,20-trione, 6a,9a,2ltrifiuoro-l6a, l7a-dihydroxy-4-pregnene-3,1 1,20-trione, 6a,21-difluoro-l 6oz, l7a-dihydroxy-1,4-pregnadiene-3,l l,20-trione, and6a,9a,21-trifl11oro-l6a,17a-dihydroxy-1,4-prcgnadiene-3,11,20-trione.The alcohols are obtained from the acylates by hydrolysis in accordancewith general hydrolysis procedures known in the art. A preferredprocedure is to employ at least a molar equivalent of an alkali-metalbicarbonate in a substantially oxygen-free solution of a mixture of alower alkanol and water. The hydrolysis reaction is carried out at atemperature between ten and thirty degrees centigrade while protectingthe mixture from atmospheric oxygen. After the hydrolysis is complete,the reaction mixture is neutralized with an acid, e. g., acetic acid,and the hydrolyzed prod uct recovered from the reaction mixture byevaporation and crystallization, extraction with methylene chloride, orthe like. The 16-esters when desired, can again be prepared byesterification of the hydroxyls by esterification procedureshereinbefore described. The preferred esters are those derived from anorganic carboxylic acid, preferably a hydrocarbon carboxylic acidcontaining from one to twelve carbon atoms, inclusive.

The following preparations and examples are illustrative of the processand products of the present invention but are not to be construed aslimiting.

PREPARATION l The 3-ethylene ketal of methyl3,11-di/ccl0-5a,6aoxido-I7(20) -[cis] -pregnen-21-0ate To a solution of5.0 grams of the 3-ethylene ketal of methyl3,11-diketo-4,17(20)-[cis]-pregnadien-21-oate, prepared in the mannerdescribed in U. S. Patent 2,707,- 184, in milliliters of chloroform wasadded a chilled solution of 1.9 grams of perbenzoic acid dissolved in31.5 milliliters of chloroform. The solution was maintained at aboutfour degrees centigrade for 24 hours, and

then at room temperature for 72 hours. The solution was then washed witha five percent aqueous solution of sodium bicarbonate and then withwater. The chloroform layer was separated, dried and the solventdistilled to give a residue of 5.3 grams of solid. Crystallization ofthis solid from methanol gave 2.24 grams of product melting at todegrees centigrade and after two crystallizations from methanol, therewas obtained pure 3-ethylene ketal of methyl 3,11-diktO,5a,6a-OXid0-17(20)-[cis]-pregnen-2loate melting at 206 to 209 degrees Found: C, 69.59;H, 7.81.

PREPARATION 2 Methyl 3,1 1-diketo-5a-hydroxy-6/3-fluoro-17(20)-allopregnen-ZI-oate Centigrade having an'iuJ of plus 37 degrees CI -ICITo a solution of 1.73 grams of 3-ethylene ketal of I methyl 3 11-diketo-5a,6a-oxido-17 20 cis -pregnen-21- oate in sixteen millilitersof methylene chloride was added sixmilliliters of 48 percenthydrofluoric acid. The heterogeneous mixture was stirred fortwo hours,made slightly basic with 300 milliliters of five percent sodiumbicarbonate solution, and extracted with methylene chloride. The extractwas washed, dried and evaporated to dryness to give 1.62 grams of crudesolid. Chromatography gave two fractions: A, 481 milligrams eluted withmethylene chloride plus five percent acetone and B, 921 milligramseluted with methylene chloride plus ten, and twenty percent acetone.Crystallization of fraction A from acetone-Skellysolve B hexanes gave390 milligrams of methyl 3,11-diketo-5a-hydroxy-6fi-fluoro-17(20)-allopregnen-21-oate, melting point254 to 260 degrees centigrade. An analytical sample melted at 260 to 263degrees centigrade.

Analysis.-Calculated for C H O F: F, 4.84. Found: F, 4.47. l

' PREPARATION 3 Methyl 3,11-diket-5d-hydroxy-6fl-fluoro-17(20)allopregnen-ZI-oate 3-ethylene ketal A mixture of 1.9 grams of methyl3,11-diketo-5a-hy- PREPARATION 4 To a solution of 1.96 grams of methyl3,11-diketo-5ahydroxy-6 3-fluoro- 17 20) -allopregnen-2 1-oate 3-ethylene ketal in 850 milliliters of anhydrous ether was added 3.7grams of lithium aluminum hydride and the mixture was stirred for aperiod of one hour. 200 milliliters of water was added slowly and theether phase separated. The aqueous phase was extracted with ethylacetate and the extracts added to the ether phase. The combinedetherethyl acetate solution was washed with water, dried and evaporatedto dryness under reduced pressure. The crude solid residue wascrystallized from acetone-Skellysolve B hexanes to give 1.30 grams of5u,11;8,21-trihydroxy-6B- fiuoro-17(20)-allopregnen-3-one 3-ethyleneketal, melting point 197 to 205 degrees centigrade. An additional 226milligrams was obtained from the mother liquor, melting point 175 to 185degrees centigrade.

PREPARATION 5 5 0a,] 1 fi-dihydroxy-6 fi-fluoro-ZI -acet0xy-1 7 (20)allopregnen-S-one 3-ethylene ketal The acetate was prepared by allowing0.87 gram of 50.,1 15,2 1-trihydroxy-6B-fluoro- 17 (20 -allopregnen-3-one 3-ethylene ketal to stand overnight in ten milliliters of aceticanhydride and ten milliliters of pyridine. The

6 solution was then poured into ice water to give 0.92 gram of 5 a, 11fl-dihydroxy-6/3-fluoro-21-acetoxy-17(20)-allopregnen-3-one 3-ethyleneketal, melting point to degrees centigrade, which on recrystallizationfrom acetone-Skellysolve B hexanes gave 0.77 gram, melting point 149 to153 degrees centigrade.

PREPARATION 6 To a solution of 0.77 gram of 5a,11;3-dihydroxy-6B- fiuoro21 acetoxy-17(20)-allopregnen-3-one 3-ethylene ketal in 35 millilitersof tertiary butyl alcohol was added one milliliter of pyridine, 1.9milliliters of 1.95 molar tetiary butyl alcohol solution ofN-methylmorpholine oxide peroxide, and 13.1 milligrams of osmiumtetroxide (9.1 milliliters of tertiary butyl alcohol solution containing1.44 milligrams OsO per milliliter). The solution was stirred for aperiod of 2.5 hours, fifteen milliliters of five percent sodiumhydrosulfate added, stirred for an additional ten minutes, 0.7 gramoffinely ground synthetic magnesium silicate added, stirred for a periodof twenty minutes more and filtered. The filtrate was taken to drynessunder reduced pressure (below fifty degrees centigrade) and the residuedissolved in methylene chloride, washed with water, dried and evaporatedto dryness. This residue was crystallized from acetone-Skellysolve Bhexanes to give 0.47 gram of 5a,- 11/3,17a-trihydroxy-6fi-fiuoro 21acetoxyallopregnane- 3,20-dione 3-ethylene ketal, melting point 220 to228 degrees centigrade.

PREPARATION 7 5a,] 113,1 7ot-trihydroxy6,8-fluoro-21acetoxyallopregnane- 3,20-dione A solution of 0.47 gram of5a,11B,17a-trihydroxy-6B- fluoro-21-acetoxyallopregnane 3,20 dione3-ethylene ketal in 35 milliliters of acetone and four milliliters ofone normal sulfuric acid solution was gently boiled on the steam bathfor ten minutes, cooled and neutralized with dilute sodium bicarbonatesolution. Addition of water and cooling gave 0.33 gram of5a,11/3,17a-trihydroxy-6B-fluoro 21 acetoxyallopregnane 3,20 dione,melting point 230 to 240 degrees centigrade.

PREPARATION 8 A solution of 100 milligrams of 5a,11fi,17u-trihydroxy-6fi-fluoro-2l-acetoxyallopregnane 3,20 dione in 4.9 milliliters ofacetic acid and 0.1 milliliter of water was refluxed for a period of onehour, cooled, diluted with fifty milliliters of water and evaporated todryness under reduced pressure. The residue was chromatographed overFlorisil (synthetic magnesium silicate) to give one fraction (77milligrams) eluted with methylene chloride plus ten percent acetone.Crystallization from acetone- Skellysolve B hexanes gave 38 milligramsof Git-fluoro- 11B,17a-dihydroxy2l-acetoxy-4-pregnene 3,20 dione(GB-fluorohydrocortisone acetate), melting point 210 to 218 degreescentigrade. Infrared data and ultraviolet data were found to be inagreement with the structure.

PREPARATION 9 Isomerization of 6 8- to 6a-fluorohydrocortisone acetate Asolution of 0.132 gram of 6B-fluorohydrocortisone acetate in twelvemilliliters of chloroform and 0.1 milliliter of absolute alcohol wascooled to minus ten degrees centigrade in an ice-salt bath and a streamof anhydrous hydrochloric acid was gently bubbled through the solutionfor 2.5 hours while the temperature was maintained between minus fiveand minus fifteen degrees centigrade. The solution was then diluted with25 milliliters of chloroform, washed with dilute sodium bicarbonate andwater, dried over anhydrous sodium sulfate, and evaporated to drynessunder reduced pressure at sixty degrees centigrade or less.Crystallization of the residue from acetone-Skellysolve B gave 42milligrams of product, 6e-fluorohydrocortisone acetate, melting point203 to 210 degrees centigrade.

PREPARATION 10 dione (6a-fluorohydrocortisone) A solution of 1.1 gramsof 6a-fiuorohydrccortisone acetate, 1.0 gram of potassium bicarbonate,100 milliliters of methanol and fifteen milliliters of water was purgedwith nitrogen and stirred at 25 degrees centigrade for four hours. Thesolution was then neutralized by addition of acetic acid and themethanol was removed by distillation under reduced pressure. The residuewas extracted with 100 milliliters of methylene dichloride and theextract, after drying over sodium sulfate, was

chromatographed over a column of eighty grams of synthetic magnesiumsilicate. The product fraction was eluted with Skellysolve B hexanesplus twenty and thirty percent acetone and gave 770 milligrams of6a-fluorohydrocortisone which melted at 192 to 195 degrees centigradeafter crystallization from ethyl acetate-Skellysolve B hexanes. Ananalytical sample melted at 192 to 201 degrees centigrade and had arotation of [:1 plus 127 degrees (chloroform).

Analysis.-Calculated for C I-1 0 1 C, 66.29; H, 7.68; F, 4.99. Found: C,66.28; H, 7.65; F, 4.43.

PREPARATION 11 I -dehydro-6a-flu0r0hydr0c0rtisone Five 100-milliliterportions of a medium in 250-milliliter Erlenmeyer fiasks, containing onepercent glucose, two percent corn steep liquor (sixty percent solids)and tap water, were adjusted to a pH of 4.9. This medium was sterilizedfor 45 minutes at fifteen pounds per square inch pressure and inoculatedwith a one to two day growth of Septomyxa afiinis, A. T. C. C. 6737. TheErlenmeyer flask was shaken at room temperature (about 26 to 28 degreescentigrade) for a period of three days. At the end of this period thisSOD-milliliter volume was used as an inoculum for ten liters of the sameglucose-corn steep liquor medium which, in addition, contained tenmilliliters of an antifoam compound (a mixture of lard oil andoctadecanol). The fermentor was placed into the water-bath, adjusted to28 degrees centigrade and the contents stirred (300 R. P. M.) andaerated (0.3 liter air per minute to five liters of beer). incubation,when a good growth had been developed, five grams of6u-fluorohydrocortisone acetate plus onehalf gram of3-ketobisnor-4-cholen-22-al, dissolved in milliliters ofdimethylformamide was added and the incubation carried out at the sametemperature (28 degrees centigrade) and aeration for a period of 72hours (final pH 8.3). The mycelium was filtered 01f and washed withwater. The wash water was combined with the filtrate and the whole wasextracted with three two-liter portions of a mixture of methylenechloride ethyl acetate (3:1). Removal of the solvent by evaporation gave5.25 grams of crude solid which was triturated twice with fourmilliliters of methylene chloride to give 2.4 grams of1-dehydro-6o-fiuorohydrocortisone of melting point 198 to 203 degreescentigrade. An analytical sample, recrystallized from acetone, melted at202 to 204 degrees centigrade. Analysis gave [od plus 73 degrees(dioxane) and the following:

Analysis.-Calculated for C21H2705F: C, 66.65; H, 7.10; F, 5.02. Found:C, 66.69; H, 7.19; F, 5.49.

PREPARATION 12 6oz fluoro 17a,21 dihydroxy 4,9(11) pregnadiene-3,20-di0ne 21-acetate To a solutionof one gram of6a-fiuorohydrocortisone After 24 hours of acetate in ten milliliters vofpyridine was added 0.4 gram of N-bromoacetamide. The mixture was allowedto stand under nitrogen for twenty minutes, at which time it was cooledto five degrees centigrade. -While stirring, anhydrous sulfur dioxidewas passed over the surface until the solution gave no color change withacidified starchiodide paper. The-temperature of the reaction mixturewas not allowed to go above twenty degrees centigrade during the sulfurdioxide addition. The mixture was then allowed'to stand for fiveminutes'and was poured into 100 milliliters of ice-water, resulting inprecipitation of 915 milligrams of crude solid, melting point 190 to 202degrees centigrade. Crystallization from acetone gave 511 milligrams of6ot-fluoro-17a,2l1-dih,ydroxy-4,*9( 1 l )-pregnadiene-3,20-dione21-acetate, melting point 214 to 218 degrees centigrade. Ananalyticalsample melted at 220 to 227 degrees centigrade. Analysis gave [04], plus73 degrees (acetone) and the following:

Analysis.-Calculated for C H O C, 68.30; H, 7.23; F, 4.70. Found: C,68.77;H, 7.57; F, 4.77.

PREPARATION 13 6a fluoro 90c bromo I1fi,17a,21 trihydroxy 4- prcgnene3,20 dione 21 acetate fluoro 9abromo-hydrocortisone acetate) To asolution of 420 milligrams of 6u-flu01'0-17a,21- dihydroxy-4,9( 1 1pregnadiene-lZO-dione 21-acetate in 6.5 milliliters of methylenechloride was added 12.5 milliliters of tertiary butyl. alcohol, ,asolution of 1.0 milliliter of 72 percent perchloric acid in 75milliliters of water, and a solution of 182 milligrams ofN-bromoacetamide in 3.2 milliliters of tertiary butyl alcohol. Afterstirring for fifteen minutes, a solution of 182 milligrams of sodiumsulfite in ten milliliters of water was added and the mixtureconcentrated under reduced pressure at sixty degree centigradeuntilcrystallization occurred. After cooling in an ice bath, thirtymilliliters of water Was added with stirring. The crystalline productwas filtered, washed with water and dried, giving a yield of 480milligrams of essentially pure 6ot-fiuoro-9a-bromo-11B,l7a,21-trihydroxy-4-pregnene-3,20-dione 21-acetate, melting point 163 to 166 degreecentigrade (with decomposition). The product can be used in thesucceeding example without further purification.

PREPARATION 14 6oz flucro 95,115 oxido 170:,21 dihydroxy 4-pregnene-3,20-di0ne 21 -acetate A mixture of 2.816 grams of6ot-fiUOIO-9a-b1'OIIIO-1l/3, 17,21 trihydroxy 4 pregnene 3,20 dione 21acetate (6a-fluoro-9u-bromohydrocortisone acetate), 2816 grams ofpotassium acetate, and ninety milliliters of acetone was stirred andheated at reflux temperature for eighteen hours. The reaction mixturewas then concentrated to about one-half of the original volume andcooled in an ice-bath. Addition of 250 milliliters of water gave 2.264grams of 6a-fluoro-9/3,11fi-oxido-l7a,21-dihydroxy-4-pregnene-3,20-dione 2l-acetate, melting point to 200 degreescentigrade with decomposition. The analytical sample, recrystallizedfrom acetone, melted at 197 to 200 degrees centigrade. Analysisgave [a]plus 28 degrees (acetone) and the following:

Analysis.-Calculated for C2 H O F: C, 65.70; H, 6.95; F, 4.52. Found: C,65.76; H, 7.03; F, 4.24.

PREPARATION 15 6a,9a difluoro 1],8,I7a,21 trihydroxy 4 pregnene-3,20-di0ne ZI-acetate (60:,9a-difluorohydrocortisone acetate) To 3.41grams ofliquid hydrogen fluoride cooled in a Dry-Ice bath wasadded,portion-wise, a slurry of 1.875 grams of .6ot-fluoroe9fl,llfi-oxido-l7a,2l-dihydroxy-4- pregnene-3,20-dione ZI-acetate in 5.97grams of tetrahydrofuran, ,(distilled over sodium hydroxide) and twentymilliliters of methylene chloride which had similarly been cooled in aDry-Ice bath. The steroid dissolved completely. After standing at zeroto five degrees centigrade for seventeen hours, the reaction mixture waspoured-slowly into a stirred mixture of 300 milliliters ice-water, fiftymilliliters of methylene chloride, and twenty grams of sodiumbicarbonate. The mixture was stirred for a few minutes, the methylenechloride layer was separated and the water phase extracted with twofifty-milliliter portions of fresh methylene chloride. The combinedmethylene chloride solutions were washed with water and dried. Attemptsto crystallize the product by addition of Skellysolve Bhexanes gave onlyoil. The oil was again dissolved by addition of methylene chloride andchromatographed over synthetic magnesium silicate. fractionzof 1.496grams of crystalline product came down from the column, which, accordingto papergram analysis, was a mixture of several components.

The whole column fraction was acetylated overnight with ten millilitersof acetic anhydride in ten milliliters of pyridine. The acetylationmixture was'poured into ice-water and extracted with methylene chloride.The extract was washed with dilute acid, dilute base, water, dried andput over a synthetic magnesium silicate column. The fraction (1.075grams) eluted from the column with fifteen, and twenty percent acetonein Skellysolve B hexanes, was recrystallized several times and gave 180milligrams of constant melting product, 6a,9u-difiuorohydro cortisoneacetate, melting point 220 to 225 degrees centigrade. Analysis gave [alplus 115 degrees (acetone) and the following:

Analysis.Calculated for C H O F C, 62.71; H, 6.87; F, 8.63. Found: C,62.85; H, 7.22; F, 8.67.

PREPARATION 16 Nitrogen was bubbled through a solution of 0.33 gram of6a,9a-difluoro-11p,17u,21-trihydroXy-4-pregnene-3,20- dione 21-acetate(6ot,9ot-difluorohydrocortisone acetate) in 33 milliliters of methanolfor fifteen minutes. To this was added a solution of 0.33 gram ofpotassium bicarbonate in four milliliters of water, likewise treatedwith nitrogen. After stirring under nitrogen for five hours, the basewas neutralized by the addition of 2.5 milliliters of five percenthydrochloric acid. The mixture was then concentrated under reducedpressure at fifty degrees centigrade to about five milliliters. Theresidue was One long grees centigrade.

PREPARATION 1s 6pz-flu0r0-I7a-hydroxy-21-acetoxy-1,4,9 (11-pregnatriene- 3,20-dione To a solution of 1.05 grams of1-dehydro-6u-fluorohydrocortisone acetate in ten milliliters of pyridinewas added 0.517 gram of N-bromoacetamide. The mixture was allowed tostand under nitrogen for fifteen minutes, at which time it was cooled tofive degrees centigrade. While stirring, sulfur dioxide was passed overthe surface until the solution gave no color change with acidifiedstarch-iodide paper. The temperature of the reaction mixture was notallowed to go above twenty degrees centigrade during the sulfur dioxideaddition. The mixture was then poured into 100 milliliters of ice-water,resulting in precipitation of 977 milligrams of 6Dt-fll101'O-1706-hydroxy-2Jl-acetoxy-1,4,9( 1 1 -pregnatriene 43,20 dione, melting point186 to 196 degrees centigrade (with decomposition). An analytical samplemelted at 213 to 216 degrees centigrade (with decomposition). Analysisgave [ocl plus 34 degrees (acetone) and the following:

Analysis.Calculated for C23H27O5FZ C, 68.64; H, 6.76; F, 4.72. Found: C,68.85; H, 6.86; F, 4.72.

PREPARATION 19 taken up in ethyl acetate, washed with water, dried andPREPARATION 17 6a -fluoro 1113,17oc dihydroxy-Zl-acetoxy1,4-pregnadiene-3,20-di0ne (1-dehydr0-6ot fluorohydrocortisone acetate)A solution of two grams of 1-dehydro-6ot-fluorohydrocortisone in tenmilliliters of pyridine and ten milliliters of acetic anhydride wasallowed to stand at room temperature for seventeen hours, and was thenpoured into a mixture of ice and water. The resulting crystallineproduct was isolated by filtration, washed with water and dried- Theyield of 1-dehydro-6a-fiuorohydrocortisone a'cetoxy 1,4 pregnadiene 3,20dione (1 dehydro- 6a-flu0r0-9ot-br0m0hydrocortisone acetate) To asolution of 1.27 grams of 6a-fluoro-17a-hydroxy- 21 acetoxy 1,4,9(11)pregnatriene 3,20 dione in 19.5 milliliters of methylene chloride wasadded 38 milliliters of tertiary butyl alcohol, a solution of threemilliliters of 72 percent perchloric acid in 22.5 milliliters of Water,and a solution of 0.55 gram of N-brornoacetamide in 9.6 milliliters oftertiary butyl alcohol. After stirring for fifteen minutes,'a solutionof 0.55 gram of sodium sulfite in thirty milliliters of water was addedand the mixture concentrated under reduced pressure at sixty degreescentigrade until crystallization occurred. After cooling in an ice bath,milliliters of water was added with stirring. On filtering thecrystalline product, followed by washing with water and drying, a yieldof 1.59 grams of essentially pure 6ot-fi1l010-9a-bl0fl'lO-ll3,17cc-dihydroxy-Zl-acetoxy-1,4-pregnadiene-3,ZO-dione, melting point188 to 191 degrees centigrade (with decomposition) was obtained. Theproduct can be used in the succeeding example without furtherpurification.

PREPARATION 20 60c fluoro 9;3,11B oxia'o 17a hydroxy 21 acetoxy- 1,4pregnadiene-3,20-dione A mixture of 1.749 grams of6mfluoro-9m-bromo-1lfl, 17a -,dihydroxy 21 acetoxy 1,4 pregnadiene 3,20-dione (1-dehydro-6a-fiuoro-9a-bromohydrocortisone acetate) 1.749 gramsof potassium acetate, and fifty milliliters of acetone was stirred andheated at reflux temperature for eighteen hours. The reaction mixturewas then concentrated to about one-half the original volume cooled andpoured into 300 milliliters of water to give 1.303 grams of6a-fluoro-9B,11fi-oxido-17a-hydroxy-21-acetoxy-l,4-pregnadiene-3,20-dione, melting point 234 to 238 degreescentigrade (with decomposition). An analtical sample, recrystallizedfrom acetone, melted at 257 to 260 degrees centigrade. Analysis gave [alplus seventy degrees (acetone) and the following:

Analysis.Calculated for C H O F: C, 66.01; H, 6.50; F, 4.54. Found: C,65.73; H, 6.58; F, 3.87.

' 1"1 PREPARATION 21 604,90: difluoro 115,170: dihydroxy 21 acetoxy 1,4-

pregnadiene 3,20 dione (1 dehydro- 6a,9a difluorohydrocortisone acetate)To 5.2 grams of, liquid hydrogenfluoride cooled in a Dry-Ice bath, wasadded, portion-wise, a slurry of 2.276 grams of6u-fluoro-9631lB-oxidod7a-hydroxy-21-acetoxyl,4-pregnadiene-3,20-dionein nine grams of tetrahydrofuran (distilled over NaOI-I) and 28milliliters of methylene chloride which had similarly been cooled in aDry-Ice bath. The steroid dissolved completely. After standing at zeroto five degrees centigrade for seventeen hours, the reaction mixture waspoured slowly into a stirred mixture of 500 milliliters of water and 25grams of sodium bicarbonate. The mixture was stirred for a few minutes,and the product was extracted with three 100-milliliter portions ofmethylene chloride. The methylene chloride solutions were washed withwater, dried, and chromatographed over synthetic magnesium silicate. Thefraction eluted from the column with fifteen and twenty percent acetonein Skellysolve B hexanes was recrystallized from ethylacetate-Skellysolve B hexanes and gave 1.342 grams of1-dehydro-6a,9wdifluorohydrocortisone acetate, melting point 238 to 242degrees centigrade. An analytical sample melted at 239 to 242 degreescentigrade. Analysis gave [1x1 plus 91 degrees (acetone) and thefollowing:

Analysis-Calculated for C H O F C, 63.00; H, 6.44; F, 8.67. Found: C,63.23; H, 6.82; F, 8.14.

PREPARATION 22 60;,911 diflllOiO -I1,B,17u,21 trihydroxy 1,4 pregnadiene3,20 dione (1-dehydr0-6a,9a diflur0hydr0- cortisone) Nitrogen wasbubbled through a solution of 1.4 grams of 60:,9a difluoro 115,17dihydroxy 21 acetoxy- 1,4 pregnadiene 3,20 dione (1 dehydro 60,9ocdifiuorohydrocortisone acetate) in 140 milliliters of methanol forfifteen minutes. To this was added a solution of 1.4 grams of potassiumbicarbonate in 17.5 milliliters of water likewise treated with nitrogen.After stirring under nitrogen for five hours, the base was neutralizedby the addition of 1.5 milliliters of acetic acid in forty millilitersof water. The mixture was then concentrated under reduced pressure at 55degrees centigrade until crystallization started. The slurry was thencooled in an ice bath, diluted with 100 milliliters of water, andfiltered to give 0.892 gram of 6u,9ot-difluoro-l1fi,17ot,21- trihydroxy1,4 pregnadiene 3,20 dione (1 dehydro-6oz,904-ClifiUOYOhYdIOCOItiSOI16), melting point 232 to 242degreescentigrade (with decomposition). An analytical sample melted at250 to 257 degrees centigrade (with decomposition). 257 degreescentigrade (with decomposition). Analysis gave [@1 plus 84 degrees(acetone) and the following:

Analysis.Calculated for C i-1 0 1 C, 63.62; H, 6.61; F, 9.59. Found: C,62.26; H, 7.10; F, 9.41.

PREPARATION 23 difluoro 11,B,17a,21 trihydroxy 4 pregnene- 3,20-di0ne 21-methalnesulf0nate An analytical sample melted at 250 to 12 PREPARATION24 6a,9a difluoro 21 iodo 11 8,]7a dihydroxy 4- p regnene-3,20-dione Toa solution of 0.65 gram of 6a,9a-difluoro-11B,17u,- 21 trihydroxy4-pregnene-3,20-dione 21-methanesulfonate in 65 milliliters of acetonewas added a solution of 0.65 gram of sodium iodide in seven millilitersof acetone. The mixture was allowed to reflux on the steam bath forabout ten minutes and was then concentrated to about ten milliliters anddiluted with about milliliters of water. The resulting solid 21-iodidewas isolated by filtration after cooling for a short period at zero tofive degrees centigrade. The yield was 0.57 gram of melting point todegrees centigrade (with decomposition).

PREPARATION 25 6ot,9a,21 trifluoro 116,170 dihydroxy 4 pregnene 3,20dione (6oc,9oz,21 trifluo ro 21 desoxyhydrocortisone) To a solution of0.57 gram of 6a,9u-difluoro-21-iodo-115,17adihydroxy-4-pregnene-3,20-dione in 55 milliliters of acetonitrileat fifty to sixty degrees centigrade in the dark, was added 0.4milliliter of fifty percent aqueous solution of silver fluoride.Addition was done in three equal portions at one-half hour intervals.After maintaining at this temperature for a total of one and onehalfhours, the temperature was lowered to forty to fifty degrees centigradefor an additional two and one-half hours. The solvent was then removedunder reduced pressure at fifty degrees centigrade and the black residuewas digested with three fifty-milliliter portions of acetone. Theproduct contained, in the acetone solution, was purified bychromatography over a column of synthetic magnesium silicate andcrystallization from acetone-Skellysolve B hexanes to give6a,9x,2l-trifluoro- 11B,17a-dihydroxy-4-pregnene-3,20-dione having amelting point of 210 to 235 degrees centigrade.

PREPARATION 26 6:1,9a difluoro 11,6,17a,21 trihydroxy 1,4pregnadiene-3,20-di0ne 21 -metha'nesulf0nare To a solution of 0.925 gramof 6d,90t-d1fil1OTO-11}8,17tl,- 21 trihydroxy 1,4-pregnadiene-3,20-dionein ten milliliters of pyridine previously cooled to zero to five degreescentigrade was added 0.9 milliliter of methanesulfonyl chloride. Thereaction mixture was stirred at zero to five degrees centigrade forseventeen hours, and was then poured into 100 milliliters of cold fivepercent hydrochloric acid to precipitate the solid mesylate. Theproduct, after filtration, weighed 0.832 gram, and had a melting pointof 157 degrees centigrade (with decomposition). This was used in thenext step without further purification.

PREPARATION 27 6a,90c,21 trifluoro 11,13,17oc dihydroxy 1,4pregnad'iene-iZO-dione (1 -d'ehydr0-6a,9a,21-trifluoro-21-desoxy it y dr0 cortisone A mixture of 0.5 gram of 6ot,9a-difluoro-11fi,17a,21-trihydroxy 1,4-pregnadiene-3,20-dione 2l-methanesulfonate and 0.37 gramof potassium fluoride in ten milliliters of dimethylsulfoxide wasstirred and heated on a steam bath for seventeen hours. The reactionmixture was then cooled, poured into 150 milliliters of water, andextracted with four 100-milliliter fractions of ethyl acetate. Afterdrying over sodium sulfate, the ethyl acetate solution was evaporated todryness and the residue (452 milligrams) was purified by chromatographyover a column of synthetic magnesium silicate and crystallization fromacetone-Skellysolve B hexanes to give 63 milligrams of crystals of6a,9ct,21-trifluoro-l1 3,17a-dihydroxy-1,4- pregnadiene-3,20-dione ofmelting point 267 to 272 de- An analytical melted at 273 10: C H O F F,14.31.

PREPARATION 28 611- fluoro 11B,17a,21 trz'hydroxy 4 pregnene 3,20

dione 21 -methanesulfnate To a Solution of 770 milligrams of crude6u-fiuorol1 3,17o,21-trihydroxy-4-pregnene-3,20-dione in ten millilitersof pyridine previously cooled to zero to five degrees centigrade Wasadded 0.7 milliliter of methanesulfonyl chloride. The reaction mixturewas stirred in an ice-water bath for four hours. Dilution with 100milliliters of five percent hydrochloric acid precipitated thecrystalline mesylate. The product, after filtration, weighed 900milligrams and melted at 189 to 192 degrees (with decomposition).Infrared analysis in mineral oil mull showed absorptions as follows:3560, 3420 centimeters- (OH); 1725 centimeters (20-ketone); 1655centimeters" (A -3-ketone); 1640, 1617 centimeters- (C=C); 1350, 1200,1170 centimeters (OSO PREPARATION 29 60:,21 difluoro 11 ,170. dihya'roxy4 pregnene 3,20 dione (6a,21-diflu0r0-21desoxyhydrocortisone) With fiftymilliliters of methylene chloride and washed three times withtenmilliliters of water. After drying over sodium sulfate, the methylenechloride solution was passed over a column of ten grams of syntheticmagnesium silicate. Elution with Skellysolve B hexanes plus nine percentacetone (four fractions of twenty milliliters each) gave 28 milligramsof crude crystals identified by infrared data as 60: fluoro1LB-hydroxy-17,20-epoxy-4- pregnen-3-one. The infrared adsorptionmaximums in mineral oil mull were as follows: 3410 centimeters- (OH);1807 centimeterr (C=O, 4-membered ring); 1660 centimeters- (A -3-keto);1625 centimeters (C=C).

Further elution with Skellysolve B hexanes plus twelve and fifteenpercent acetone yielded 45 milligrams of 6a,- 21 difiuoro 115,170dihydroxy 4 pregnene 3,20 dione which on recrystallization from ethylacetate Skellysolve B gave 29 mgs. melting point226 to 230 degrees.centigrade. Infrared adsorption showed the following maximums: 3600,3540, 3360 eentimeterv (OH); 1722 centimeters (20-ketone); 1653centimeters- (A -3- ketone); 1625 centimeters-- (C=C).

PREPARATION 30 6 a-fiuoro-l 1,8,1 7u,2Z-trihydr0xy-1,4-pregnadiene- 3 ,2O-dione 21 -methanesulf nate Three hundred milligrams of 6a-fiuoro11/3,17a,21-trihydroxy-1,4-pregnadiene-3,20-dione was dissolved inpyridine and cooled to a temperature of between zero and five degreescentigrade. 0.1 milliliter of methanesulfonyl chloride was added and thesolution maintained between zero and five degrees centigrade for twohours. This was poured into a solution of three milliliters ofconcentrated hydrochloric acid diluted with fifty milliliters of water.310 milligrams of crystalline 6a-fluoro- 11,13,170u21 trihydroxy 1,4pregnadiene 3,20 dione ZI-methanesulfonate was precipitated from thediluted hydrochloric acid. The product melted with decomposition at 200to 202 degrees Centigrade. Infrared adsorption, in a mineral oil mullwere as follows: 3570, 3370 centimeterr (OH), 1727 centimeters-(ZO-ketone);

I4 1665 centimeterr (conjugated ketone); 1623, i centimeterv (A 1360,1342; 1172 centimeters (OSO2).

PREPARATION 31 A mixture of 100 milligrams of 6a-fluoro-11fl,17a,21-trihydroxy-l,4-pregnadiene-3,20-dione ZI-methanesulfonate and fiftymilligrams of potassium fluoride in one milliliter of dimethylsulfoxidewas refluxed on a steam bath for eighteen hours. The mixture was dilutedwith fifty milliliters of methylene dichloride and washed two times withten milliliters of water. The methylene chloride solution was passedover a column of twenty grams of Florosil (synthetic magnesiumsilicate). Elution with a mixture composed of 91 parts of Skellysolve B(hexane hydrocarbons) and 9 parts of acetone gave a fraction of fourteenmilligrams. The infrared absorption of this compound indicated it was60L-fil101'0-1lB-llYClTOXY-170t,2loxido-1,4-pregnadiene-3,20-dione. Theabsorptions in mineral oil mull were as follows: 3380 centimeters- (OH);1807 centimeters- (four membered ring).

The fraction containing the principal product was eluted with a mixturecomposed of Skellysolve B and twelve to fifteen percent of acetone. Theproduct weighed 28 milligrams and after recrystallization from a mixtureof ethyl acetate and Skellysolve B yielded sixteen milligrams. Themelting point of the purified6m,2l-CllflLlOrO-1lfi,l7adihydroxy-1,4-pregnadiene-3,20-dione was 226 to231 degrees Centigrade. Infrared absorptions in mineral oil mull follow:3360 centimeters- (OH); 1725 centimeters- (ketone); 5 centimeters"(conjugated ketone); 1597 centimeters (A EXAMPLE 1 6a,21-diflu0r0-11,8,16a,1 7 a-trihydroxy- -pregnene- 3,20-di0ne One hundred millilitersof two percent corn steep liquor of sixty percent solids was adjusted topH of 6.8 i

to 7.4 with sodium hydroxide and was sterilized at fifteen poundspressure for thirty minutes. To this was added a similarly sterilesolution of two grams of Cerelose (technical grade of dextrose) in fourmilliliters of water. This sterile medium was inoculated with asuspension of spores and mycelium of Streptomyces roseochromogenus(Waksman Collection No. 3689) and was agitated on a rotatory shaker fora period of 24 hours by which time a good growth of the organism hadtaken place. To this 24 hour culture, twenty milligrams of60,21-difluoro-1 16,17a-dihydroxy-4-pregnene-3,20-dione dissolved in 0.2milliliter of dimethylformamide was added. Incubation of the steroidwith the microorganism was maintained (with agitation) for five days, atwhich time the pH was 8.6. The fermentation broth was then separatedinto the mycelium and the beer by centrifugation. The mycelium wasextracted. first with two 25 milliliter portions of acetone and thenwith four successive 25 milliliter portions of methyl'isopropyl ketone.The beer was extracted with four successive 25 milliliter portions ofmethyl isopropyl ketone. All of the extracts were combined, washed withtwo percent aqueous sodium bicarbonate solution and with water, driedwith anhydrous sodium sulfate, and evaporated to dryness. The residue,which on paper chromatogram analysis showed the presence of6e,2l-difluoro-1lfl,l6a,l7a-trihydroxy- 4-pregnene-3,20-dione, waspurified by chromatography over synthetic magnesium silicate (Florisil)and crystallization from acetone to give6a,2l-difluoro-11fl,16u,l7mtrihydroxy-4-pregnene-3,20-dione.

Following the'procedure of Example 1 above, but substitutin g6a,9a,21-trifluoro-1 1,8, l7u-dihydroxy-4-pregnene- 3,20-dione,6oz,21-difll1OIO-1 1,8, l7a-dihydroxy-1,4-pregnadiene-3,20-dione, or6a,9a,2l-trifluoro-1lfi,17e-dihydroxy- 1,4-pregnadiene-3,20-dione asstarting material therein,

is productive of 6a,9 x,21-trifiuoro-11,8,16e,17ottrihydrox4-pregnene-3,20-dione,6a,2l-difiuoro-11/3,l6ct,17a-trihydroxy-1,4-pregnadiene-3,20-dione, and6a,9c:,2l-t11fit1010- l1B,l6ot,17u trihydroxy 1,4 pregnadiene 3,20dione, respectively.

EXAMPLE 2 A solution of 1.2 grams of6a,21-difluoro-11,8,16o:,17tttrihydroxy-4-pregnene-3,ZO-dione (fromExample 1) in twenty milliliters of pyridine and twenty milliliters ofacetic anhydride was allowed to stand at room temperature (about degreescentigrade) for eighteen hours and was then poured into 200 millilitersof ice-water. The resulting mixture was extracted with methylenechloride and the extract was washed with dilute hydrochloric acid,dilute sodium bicarbonate and water. After drying the solution withanhydrous sodium sulfate, the solvent was removed by evaporation and theresidue was purified by chromatography over synthetic magnesium silicate(Florisil) and crystallization from acetone to give6m,21-difluoro-11p,16a,17u-trihydroxy-4-pregnene- 3,20-dio-ne16-acetate.

Following the procedure of Example 2 above but substituting6a,9u,21-trifluoro-11B,l6a,17e-trihydroxy4- pregnene-3,20-dione,60,21-dlfi't101O-l 113, 1 6a,17o-t1ihydroxy-l,4-pregnadiene-3,20-dione,or 6a,9a,2l-trifiuoro- 1l{3,16r1,l7e trihydroxy 1,4 pregnadiene 3,20dione as starting material therein is productive of 6e,9t,21-trifluoro-l1,3,16oc,17a trihydroxy 4 pregnene 3,20 dione 16-acetate, 6e,21-difiuoro11,8,16e,17a trihydroxy 1,4- pregnadiene-3,20-dione l6-acetate and6a,9ix,2l-trifiuoro- 1l{3,16ot,17a trihydroxy 1,4 pregnadiene 3,20 dionel6-acetate, respectively.

Similarly, acylation of6a,21-difiuoro-11fi,16a,17ot-trihydroxy-4-pregnene-3,20-dione with theappropriate acid anhydride or acid chloride is productive of still other16-acylates such as, for example, 6a,21-difiuoro-11,B,16a,17a-trihydroxy-4-pregnene-3,20-dione 16-propionate, 6a, 21 difluoro11,8,16ot,17t trihydroxy 4 pregnene- 3,20-dione 16-butyrate,6a,21-difluoro-1lfl,16ct,17a-trihydroxy-4-pregnene-3,ZO-dione16-valerate, 6u,2l-difluoro-11,8,16ix,17a.-trihydroxy-4-pregnene-3,20-dione 16-hexanoate,6tx,2ldifl11OIO-l131,l6a,17 0L-tIihYdIOXY-4-P16gll6fl6- 3,20-dionel6-laurate, 6a,21-di1'luoro-llB,16o,l7o-trihydroxy-4-pregnene-3,ZO-dione lo-trimethylacetate, 6u,21-difiuoro-l 1,8,16a,17w-trihydroxy-4-pregnene-3,ZO-dione l6- isobutyrate,60:,2l-Clil'lllOfO-l 15,16a,17a-tril1ydroxy-4-pregnene-3,20-dionelo-isovalerate, l6a,21-difiuoro-l1,8,16a,17ot-trihydroxy-4-pregnen-3,ZO-dione 6-cyc1ol1exane carboxylate,6e,21-difiuoro-11p,16a,17e-trihydroxy-4-pregnone-3,20-dione lo-benzoate,60:,2l-difltl0lO-llfl,l6o:,17oztrihydroxy-4-pregnene-3,20-dione16-phenylacetate, 60:, 21 difiuoro 11,8,16o,17ot trihydroxy 4 pregnene-3,20-dione 16-(B-phenyl)propionate, 6a.,Zl-dlflll0f04lfi,16a,17a-trihydroxy-4-pregnene-3,20-dione 16-(o-, m-) ptoluate, 641,21difiuoro l1fi,16ot,17a trihydroxy 4- pregnene-3,20-dione16-hemisuccinate, 602,21-(lifl11010- 11fl,16a,17utrihydroxy-4-pregnene-3,ZO-dione 16-hemiadipate, 60:,21 difiuoro1l,8,16a,l7c2 trihydroxy 4- pregnene-3,20-di0ne lo-acrylate,6m,2l-dlflUOfO-l1;3,l6oz, 17a trihydroxy 4 pregnene 3,20 dione 16undecylenate, 600,21 difiuoro l1B,l6cc,17a trihydroxy 4-pregnene-3,20-dione 16-propiolate, 6a,2l-difiuoro ll,d,16a,17a-trihydroxy-4-pregnenc-3,20dione 1 o cinnamatc, 6a,21 fiuorol1,8,l6o,17a trihydroxy 4 pregnene- 3,20-dione l6maleate,60:,2l-tliflUUIO-llfi,l7c-lfll1yd1'OXy I 4-pregnene-3,2L-dionelo-citraconate.

Similarly, acylation of 6a,9ot,2ltrifiuoro-llp,16ct,l7o:-trihydroxy-4-pregnene-3,20-dione, 6a,2l-difl1l0IO-11 3,16ot,l7e-trihydroxy-l,4-pregnadiene-3,ZO-dione or 6o:,9a,2ltrifiuoro ll;.3,la,l7c: trihydroxy 1,4 pregnadiene- 3,20-dione with the appropriateaeylating agent is productive of the corresponding l6-acylates. Thepreferred 1e acylates are those corresponding to the acylates describedabove for 6a,21-difiuoro-11,8,16a,17a-trihydroxy-4-pregnene-3,20-dione.

EXAMPLE 3 6:1,21 -diflu0r0-1 6 0a,] 7a-dihydroxy-4-pregnen e-3,11,204ri0ne 16-acetate To a solution of 0.5 gram of60:,2l-dlflll01O-ll;3,16oc, 17a-trihydroxy-4-pregnene-3,20-dione16-acetate in twenty milliliters of acetic acid was added a solution of0.15 gram of chromium trioxide in one-half milliliter of water. Themixture was stirred'and maintained at room temperature (about 25 degreescentigrade) for a period of four hours. Thereafter, the excess oxidantwas destroyed by the addition of 0.5 milliliter of methanol and themixture was poured into milliliters of water and extracted withmethylene chloride. The extract was washed with dilute sodiumbicarbonate and with water, and was dried and evaporated to dryness. Theresidue was crystallized from acetone to give6ix,21-diflu0ro-16a,Not-dihydroxy- 4-pregnene-3,11,20-trione 16-acetate.

Following the procedure of Example 3 above but substituting6a,9m,21-trifluoro-11/3,16a,17a-trihydroxy-4-pregnone-3,20-dionel6-acetate,6a,21-difluoro-1lB,l6u,17atrihydroxy-1,4-pregnadiene-3,20-dione16-acetate, 6oz,9 x, 21-trifluoro-11,8,16a,17u-trihydroxy-1,4-pregnadiene-3 ,20- dione 16-acetate, or6a,9ot,21-trifluoro-11fl,16o ,17a.-trihydroxy-l,4 pregnadiene-3,ZO-dione16-acetate as starting material therein is productive of6a,9a,Zl-trlfi1lOrO-l6a, 17a-dihydroxy-4-pregnene-3,11,20-trione16-acetate, 6a, 21 difiuoro l6ot.,17vx dihydroxy 1,4 pregnadiene-3,11,20-trione 16-acetate, and 606,905.,2l-t1lfll10lO-160!,1711-dihydroxy-1,4-pregnadiene-3,l1,20-trione '16-acetate, respectively.

Similarly oxidation of other llp-hydroxy 16-ester compounds of Example 2is productive of the corresponding ll-keto 16-ester compounds. Thepreferred ll-keto 16- ester compounds are those wherein the acyl radicalis that of an organic carboxylic acid, preferably a hydrocarboncarboxylic acid containing from one to twelve carbon atoms, inclusive.

EXAMPLE 4 A solution of 1.2 grams of6a,2l-di1lu0rO-l6o:.,l7adihydroxy-4-pregnene-3,11,20-trione l6-acetate,two grams of potassium bicarbonate, 100 milliliters of methanol andfifteen milliliters of water was purged with nitrogen and stirred at 25degrees centigrade for eight hours. The solution was then neutralized byaddition of acetic acid and the methanol was removed by distillationunder reduced pressure. The residue was extracted with 100 millilitersof methylene chloride and the extract, after drying over sodium sulfate,was c-hrornatographed over a column of eighty grams of syntheticmagnesium silicate using Skellysolve B with increasing amounts ofacetone for elution. The product fraction from the column wascrystallized from acetone to give 6o:,Z1-difiuOI0-l6rx,l7otdihydroxy-4-pregnene-3 ,1 1,20-trione.

Following the procedure of Example 4, saponification of 60,9oz,21trifluoro l6ot,17oc dihydroxy 4 pregnene-3,ll,20-trione l6-acetate,60:,21-(111111010-1611,l70t-dihydroxy-1,4-pregnadiene-3,11,20-trione16-acetate and 60a, 9a,2l-trifluoro-16a,17u-dihydroxy-1,4-pregnadiene3,11, 20-trione 16-acetate of Example 3 is productive of thecorresponding hydroxy compounds, 6cc,9oc,2l-[Ylflll0l0-16a.,17ot-dihydroxy-4-pregnene-3,11,20-trione,6a,21-dihydroxy-1,4-pregnadiene-3,11,20-trione, and6e,9x,21-trifluoro-16a,17a-dihydroxy-1,4-pregnadiene-3,11,20-trione.

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds shown and described, asobvious modifications and equivalents will be apparent to one skilled inthe art,

17 and the invention is therefore to be limited only by the scope of theappended claims.

We claim: 1. A 16-0xygenated steroid of the formula:

wherein the 1,2-carbon atom linkage is selected from the linkagesconsisting of single bond and double bond linkages, Y is selected fromthe group consisting of hydrogen and fluorine, X is selected from thegroup consisting of the carbonyl radical and the fl-hydroxymethyleneradical, and R is selected from the group consisting of hydrogen and theacyl radical of a hydrocarbon carboxylic acid containing from one totwelve carbon atoms, inclusive.

2. A l6-oxygenated pregnene of the formula:

wherein Y is selected from the group consisting of hy- 46 18 5. :,21difluoro 16a,l7oc dihydroxy 4 pregnene- 3,11,20-trione.

6. 6a,9oz,21 trifluoro 16ot,l7a dihydroxy 4 pregnene-3,1 1,20-trione.

7. 60:,21 difluoro 11/3,16oc,170c trihydroxy 4 pregnene-3,20-dione16-acetate.

8. 6a,9oc,21 trifluoro 11B,16a,17u trihydroxy 4- pregnene-3,20-dione16-acetate.

9. 60:,21 difluoro :,170: dihydroxy 4 pregnene- 3,11,20-trione16-acetate.

10. 6a,9a,21-trifluoro-16a,17a-dihydroxy-4-pregnene-3, 11,20-trione16-acetate.

11. A 16-oxygenated pregnadiene of the formula:

wherein Y is selected from the group consisting of hydrogen andfluorine, X is selected from the group consisting of the carbonylradical and the fi-hydroxymethylene radical, and R is selected from thegroup consisting of hydrogen and the acyl radical of a hydrocarboncarboxylic acid containing from one to twelve carbon atoms inclusive.

12. 6a,21 difluoro 11fi,16oc,170t trihydroxy 1,4-pregnadiene-3,20-dione.

13. 6ot,9a,21 trifluoro 11,8,l6m,17a trihydroxy 1,4-pregnadine-3,20-dione.

14. 60:,21 difluoro 160:,17u dihydroxy 1,4 pregnadiene-3,1 1,20-trione.

15. 6u,9a,21 trifluoro 16u,17u dihydroxy 1,4- pregnadiene-3,11,20-nione.

16. 6a,21 difluoro 11B,16a,17ot trihydroxy 1,4- pregnadine-3,20-dione16-acetate.

17. 6a,9oa,21 trifluoro 11 3,16oz,17u trihydroxy 1,4-pregnadiene-3,20-dione 16-acetate.

18. 604,21 difluoro 16a,17a dihydroxy 1,4 pregnadiene-3,l1,20-trione16-acetate.

19. 6oc,9ot,21 trifluoro 160a,17a dihydroxy 1,4-pregnadiene-3,11,20-trione 16-acetate.

No references cited.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.2,838,547 June 10, 1958 Barney J Magerlein at ale It is hereby certifiedthat error appears in the-printed specification of the above numberedpatent requiring correction and that the said Letters Patent should readas corrected. below.

Column 1, line '71, for "-30,20d.ione" read an 3,L 3O -=dione col mm 2,line 35, for "July 8, 952" read July 8, 1952; column (5, line 155, I fortetiar w' read i--tertiaryoolmnn 15, line 50, for "l aflil read 606 ,21line 51, for cyolohexane" read 1 CYGlOhKSXEIlG a I Signed and sealedthis lz thday of October 1958 (SEAL) Attest:

KARLH, AXLINE ROBERT C. WATSON Atte sting Oflicer Commissioner ofPatents

1. A 16-OXYGENATED STEROID OF THE FORMULA: